Kaitlyn Vance and Dr. Eric Wilson, Microbiology and Molecular Biology

Mucosal surfaces are the main portals through which pathogens enter the body of an organism. Previous research has shown that mucosal immunity effectively prevents the entry of pathogens at these surfaces, thus disrupting an infection before it begins; this immunity can be achieved by mucosal vaccination (1). The homing mechanisms of lymphocytes in the mucosal immune system, however, are not yet fully understood. This mentored research project was designed to deepen the understanding of how the mucosal immune system operates. We sought to evaluate the efficacy of mucosal vaccination in producing localized immune responses by attempting to reproducibly create a robust immune response in one select mucosal tissue. We hypothesized that by increasing the expression of CCR10 on antigen specific IgA antibody secreting cells (ASC), the immune response of antibody production would be highly concentrated in specific mucosal tissues and not evenly distributed among regional mucosal tissues or evenly throughout the entire mucosal system.

We began immunizing mice in December of 2010, following the methods outlined in the research proposal. All variations of antigen- and adjuvant-containing solutions were used. Both the sublingual and intranasal methods were used to administer the solutions. Blood was periodically collected during the month-long immunization process to check for sufficient immune responses and tissues and blood were collected 4 weeks after the initial immunization. The ELISA assay was used to determine overall IgA levels as well as antigen specific immune responses. Despite the various solutions and administration methods, no antigen specific immune responses were detected against ovalbumin or cholera toxin. After several months of immunizing and testing for responses, the project methods were reevaluated.

The major issue we encountered during this project was the inability to produce a detectable immune response in the mice. The recruitment of lymphocytes to mucosal tissues involves a cascade of molecular interactions and is not entirely understood; our protocol, however, was designed with this in mind. By immunizing with various methods and using solutions with multiple antigens, our research design accounted for a wide range of the uncontrollable factors in lymphocyte recruitment. Because an antigen-specific response was not detectable in mucosal tissues and in the serum, we were led to believe that the immunization solutions were the root of the problem. I then researched previously published papers on IgA lymphocyte recruitment and the use of 1,25-dihydroxyvitamin D3 in upregulating the expression of CCR10.

Previous research shows that 1,25-dihydroxyvitamin D3 does signal cells to increase expression of CCR10, allowing activated cells to effectively migrate to the chemokine CCL27 in the epidermis (2). However, the methods of preparing immunizations containing a vitamin D3 adjuvant in the literature differed from our methods. As shown by Enioutina, et al., the antigen solution should be prepared in an aqueous solution of aluminum hydroxide and the adjuvant should be dissolved in 95% ethanol before being added to the antigen (3). In addition, all published research into vitamin D3 as an adjuvant in the mucosal immune system of mice used a subcutaneous method of administration. Based on these findings, we have modified our methods. We are now immunizing mice subcutaneously using aluminum hydroxide to deliver our antigen, and we are incorporating the vitamin D3 adjuvant after dissolving it in 95% ethanol.

Our first group of mice immunized according to this new set of methods has shown a detectable antigen-specific response at 2 and 3 weeks after initial immunization. To further account for the complexity of the mucosal immune system, we have decided to breed the immunized mice and collect the milk and the mammary gland tissue in addition to collecting the serum and salivary glands. Previous research done by Dr. Eric Wilson shows that CCR10 plays an important role in the homing mechanisms of lymphocytes to the lactating mammary gland; therefore, if vitamin D3 upregulates CCR10 expression, a robust immune response might also be detectable in the lactating mammary gland (4). In the next week we will sacrifice this initial group of mice and evaluate the antibody content.

With our modifications, we hope to see differences in antigen-specific IgA content between the groups of mice that have received the adjuvant (1,25-dihydroxyvitamin D3) and those that have not. We will have initial results in 1-2 weeks.1

Scholarly Sources

1. Neutra, M. R., and P. A. Kozlowski. 2006. Mucosal vaccines: the promise and the challenge. Nature reviews 6:148-158.
2. Sigmundsdottir, H., J. Pan, G. F. Debes, C. Alt, A. Habtezion, D. Soler, and E. C. Butcher. 2007. DCs metabolize sunlight-induced vitamin D3 to ‘program’ T cell attraction to the epidermal chemokine CCL27. Nature immunology 8:285-293.
3. Enioutina, E. Y., D. Visic, Z. A. McGee, and R. A. Daynes. 1999. The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant. Vaccine 17:3050-3064.
4. Morteau, O., C. Gerard, B. Lu, S. Ghiran, M. Rits, Y. Fujiwara, Y. Law, K. Distelhorst, E. M. Nielsen, E. D. Hill, R. Kwan, N. H. Lazarus, E. C. Butcher, and E. Wilson. 2008. An indispensable role for the chemokine receptor CCR10 in IgA antibody-secreting cell accumulation. J Immunol 181:6309-6315.